ABSTRACT CHARACTERIZING THE FECAL MICROBIOTA AND RESISTOME OF CORVUS BRACHYRHYNCHOS (AMERICAN CROW) IN FRESNO AND DAVIS, CALIFORNIA American Crows are common across the United States, well adapted to human habitats, and congregate in large winter roosts. We aimed to characterize the bacterial community (microbiota) of the crows’ feces, with an emphasis on human pathogens. The antibiotic resistance (AR) of the bacteria was analyzed to gain insight into the role crows may play in the spread of AR genes. Through 16S rRNA gene and metagenomic sequencing, the microbiota and antibiotic resistance genes (resistome) were determined. The core microbiota (taxa found in all crows) contained Lactobacillales (22.2% relative abundance), Enterobacteriales (21.9%) and Pseudomonadales (13.2%). Among the microbiota were human pathogens including Legionella, Camplycobacter, Staphylococcus, Streptococcus, and Treponema, among others. The Fresno, California crows displayed antibiotic resistance genes for multiple drug efflux pumps, macrolide-lincosamide- streptogramin (MLS), and more. Ubiquitous, urban wildlife like the American Crow may play a role in the spread of AR pathogens to the environment and human populations. Rachel Lee Nelson August 2018 CHARACTERIZING THE FECAL MICROBIOTA AND RESISTOME OF CORVUS BRACHYRHYNCHOS (AMERICAN CROW) IN FRESNO AND DAVIS, CALIFORNIA by Rachel Lee Nelson A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology in the College of Science and Mathematics California State University, Fresno August 2018 APPROVED For the Department of Biology: We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree. Rachel Lee Nelson Thesis Author Tricia Van Laar (Chair) Biology John V. H. Constable Biology Krish Krishnan Chemistry For the University Graduate Committee: Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship. Permission to reproduce this thesis in part or in its entirety must be obtained from me. Signature of thesis author: ACKNOWLEDGMENTS Python and Linux expertise - Wesley Leach Guidance - Tricia Van Laar and committee Funding - California State University, Fresno: Graduate Net Initiative, Faculty Student Research, Graduate Research and Creative Activities CSU Council on Ocean Affairs San Joaquin Valley Chapter of the Wildlife Society TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................. vii LIST OF FIGURES ................................................................................................. ix INTRODUCTION .................................................................................................... 1 Corvus brachyrhynchos (American Crow) ....................................................... 1 Gut Microbiota .................................................................................................. 7 Antibiotic Resistance (AR) ............................................................................... 9 Aims, Hypotheses, and Significance ............................................................... 15 METHODS ............................................................................................................. 17 Field Work ...................................................................................................... 17 Experimental Design ....................................................................................... 21 DNA Extraction and Sequencing .................................................................... 23 Microbiota Analysis ........................................................................................ 24 Resistome Determination ................................................................................ 29 RESULTS ............................................................................................................... 34 American Crow Microbiota ............................................................................ 34 Diversity Analyses of Fecal Microbiota ......................................................... 47 American Crow Resistome ............................................................................. 52 DISCUSSION ......................................................................................................... 56 American Crow Microbiota ............................................................................ 56 Pathogens ........................................................................................................ 62 American Crow Resistome ............................................................................. 64 Future Directions ............................................................................................. 66 CONCLUSION ...................................................................................................... 67 vi vi Page REFERENCES ....................................................................................................... 68 APPENDICES ........................................................................................................ 84 APPENDIX A: QIIME2 SYSTEM DETAILS AND COMMANDS .................... 85 APPENDIX B: MICROBIOTA DETAILS ........................................................... 89 APPENDIX C: RESISTOME RESULTS VIA ARGS-OAP ............................... 108 LIST OF TABLES Page Table 1. FeatureTable (table.qzv) Displaying Number of Aequences Per Sample Remaining After Quality Control (DADA2). .......................................... 25 Table 2. Isolation Agars Used to Select Organisms from Fecal Samples .............. 30 Table 3. Test Antibiotics Modes of Action ............................................................ 31 Table 4. 96 Well Plate Set Up Used to Determine Minimal Inhibitory Concentrations ......................................................................................... 31 Table 5. Taxonomic Richness of Fresno, Davis, and Critter Creek Microbiota .... 34 Table 6. Relative Abundance of Phyla and Orders in the American Crow Microbiota ................................................................................................ 35 Table 7. Taxa Identified in All Fecal Samples, Comprising the Core Fecal Microbiota of the American Crow ........................................................... 40 Table 8. Fresno American Crow Core Fecal Microbiota ....................................... 41 Table 9. Critter Creek American Crow Core Fecal Microbiota ............................. 42 Table 10. Davis American Crow Core Fecal Microbiota ....................................... 43 Table 11. Fresno American Crow Variable Fecal Microbiota ............................... 44 Table 12. Critter Creek American Crow Variable Fecal Microbiota ..................... 44 Table 13. Davis American Crow Variable Fecal Microbiota ................................. 45 Table 14. Number of Pathogen-Containing Taxa in American Crow Microbiota . 46 Table 15. Summary of Microbiota Diversity Statistical Analyses ......................... 48 Table 16. Antibiotic Resistance Genes Identified in Fresno Resistome ................ 52 Table 17. Minimum Inhibitory Concentrations (µg/mL) of Critter Creek Isolates ........................................................................................... 53 Table 18. Minimal Inhibitory Concentration (µg/mL) Observed in Fresno, California Fecal Microbiota ..................................................................... 54 Table 19. Antibiotic Resistant Organisms Isolated from Fresno Crow MIC ......... 54 viii viii Page Table 20. Minimum Inhibitory Concentrations (µg/mL) of CSU, Fresno Farm Animals Isolates ....................................................................................... 55 Table 21. Full Microbiota of American Crow Feces ............................................. 90 Table 22. Complete Relative Abundance of Phyla in the Fresno Crow Microbiota .................................................................................... 105 Table 23. Complete Relative Abundance of Phyla in the Critter Creek Crow Microbiota .............................................................................................. 106 Table 24. Complete Relative Abundance of Phyla in the Davis Crow Microbiota .................................................................................... 107 Table 25. Complete List of Antibiotic Resistance Genes Identified by ARGs-OAP. ...................................................................................... 109 LIST OF FIGURES Page Figure 1. Map of American Crow staging and roosting locations in Fresno, California. ............................................................................................... 18 Figure 2. Map of American Crow staging and roosting locations in Davis, California. ............................................................................................... 20 Figure 3. Forward and reverse reads quality (Demux.qzv). ................................... 26 Figure 4. Average relative abundance of